This proposal continues studies that combine physiological and ultrastructural analysis to understand membrane dynamics and its role in vasopressin (ADH) action. These studies of the toad urinary bladder will characterize important features of the intramembrane particle aggregates and their role in the water permeability response. Utilizing a new "hyperstretch" procedure that flattens the apical membrane to allow a comprehensive evaluation of particle aggregate area, a rigorous evaluation of these structures will be carried out for conditions where previous studies indicate a dissociation between aggregates and water permeability response. These studies will also assess whether the aggregates associated with fused aggrephores and revealed by hyperstretch are important sites of water flow. Other studies will use the "composite replica" methodology,k recently developed by this laboratory, to examine the cytoplasmic membrane surface in order to establish the relationship of cytoskeletal elements to the aggregates. Membrane retrieval into the endosomal system following ADH reversal will be studied to identify the site where aggregates and fluid phase markers are sorted. By covalently labeling components on the mucosal surface with biotin, the recycling of aggregate constituents will be evaluated. Antibodies to specific aggrephore components will be localized in control and ADH-stimulated bladders using a range of sensitive protocols including a new labeled composite replica strategy. This should make it possible to determine which protein(s) relate directly to the aggregates and are likely water channel components.